During the course of the preceeding grant of the same title, we concentrated on two new techniques: 1. ribosubstitution sequencing permits one to obtain new base-specific cleavages and to use sequence specific DNases such as restriction enzymes and T4 endonuclease IV. 2. reverse transcriptase can be used to make complementary DNA copies of interesting RNA molecules so that the power of DNA sequencing techniques can be brought to bear. As part of this work we started a major effort to sequence the Gamma and Betaglobin mRNA chains from the rabbit. We will continue to develop and use these techniques but the major emphasis will be on perfection of a third new approach, deoxysubstitution sequencing, in which one synthesizes in vitro an RNA molecule in which a deoxybase replaces one of the ribonucleotides. Such heteropolymers can be cleaved specifically at several bases. For instance deoxy C substituted RNA can be cleaved at U residues by pancreatic RNase, at G residues by RNase U, and at dC residues by pancreatic DNase I. This flexibility should permit much more rapid sequencing of large molecules. BIBLIOGRAPHIC REFERENCES: Appendix VI, Salser, W., Bowen, S., Browne, D., El Adli, F., Fedoroff, N., Fry, K., Heindell, H., Paddock, G., Poon, R., Wallace, B. and Whitcome, P. (1976). "Investigation of the Organization of Mammalina Chromosomes at the DNA Sequence Level," (galley proofs will appear in Federation Proceedings, Vol. 35, January, 1976). Appendix IV, Ricard, B. and Salser, W. (1975). "The Structure of T4 Specific Messenger RNAs: Tightly Folded Conformation Revealed by Sedimentation in Denaturing Solvents." J. Mol. Biol. 94. 163.